Abstract

Depression is a major global public health concern. The variable effectiveness and undesirable side effects of antidepressant medication have prompted the search for alternative treatments. Olfaction plays an important role in emotion regulation in our daily life. Pleasant odorants would evoke positive emotions, inducing relaxation and calmness. The beneficial effects of vanillin aromatherapy on depression model rats were investigated in our previous research. Here, biochemical molecules including serum magnesium and brain derived nerve growth factor (BDNF) were further detected to investigate the possible underlying mechanisms. Results showed that the administration of vanillin aromatherapy restored the serum magnesium and brain BDNF which might mediate remission of depression. This suggests that vanillin aromatherapy could be considered as a potential alternative therapy for alleviating psychological responses among people suffering from major depressive disorder.

Keywords

Vanillin, Aromatherapy, Magnesium, Brain-derived neurotrophic factor

Introduction

Major depressive disorder (MDD) is a common psychiatric disorder affecting more than 120 million people worldwide [1]. The unsatisfactory clinical efficacy of antidepressant drugs, further compounded by a variety of adverse effects associated with current antidepressant drugs has made major depression disorder a central focus of medical research and prompted the search for novel alternative treatments.

Aromatherapy has a long history in the treatment of depression [2]. The pilot study indicates positive findings with minimal risk for the use of aromatherapy as a complementary therapy in both anxiety and depression scales with the postpartum woman [3]. Evidences also suggest that aromatherapy is effective in controlling perceived stress and depression [4,5]. Pleasant odorants can be experienced as extremely relaxing and calming, such as rose, lavender, citrus and lemon fragrance [6].

Basing on the antidepressant experience of aromatherapy, we select an odorant named vanillin to investigate its antidepressant effect on depressive model rats. In our previous research, vanillin was demonstrated to ameliorate the depression-like behaviors in depression model rats and elevated dopamine and serotonin in the brain [7]. The aim of the present study is to investigate the other possible underlying mechanisms and provide stronger evidence for alternative antidepressant effect of vanillin aroma.

Methods

▪ Animals

The present study was performed in compliance with the Animal Scientific Procedures Act of 1986 and received local ethics committee approval (license number: LLSC2013006).

Depression model rats were divided into 3 groups with 12-14 rats per group: the fluoxetine group; the vanillin group and the untreated group. Another normal 8 rats without stress stimulation were used as the control group.

Fluoxetine (10 mg/kg/d, diluted in distilled water; Eli Lilly &Co., Souzhou, China) were chronically administered to the fluoxetine group once daily for 4weeks between 8:00 and 10:00 a.m. by oral. Vanillin (Sangon Biotech, Shanghai co., Ltd, china) was administrated to the vanillin group according to the reference [7]. Rats in the untreated group and the control group received similar treatment to the vanillin group, but without any odorants administrated.

▪ Serum magnesium determination

After treatment for 4 w, 1-2 ml blood was drawn from the tail vein of anesthetized rats. After being centrifuged at 3000 rpm for 10 min at 4°C (Eppendorf, 5810R), the clear serum was obtained and sent to the laboratory department of the First Affiliated Hospital of Anhui Medical University where serum magnesium ([Mg2+]) was detected.

▪ Biological determinations

At the end of the experiment, the rats were deeply anesthetized with 10% chloral hydrate (330 μl/100 g body weight, i.p.). After transcardially perfused with physiological saline, the rats were decapitated and brains were promptly collected. The brains were cut into two equal halves from the longitudinal fissure.

One half brain was weighed and homogenized with an Ultrasonic Turax T25 (Bio-block Scientific, France) in 1 × PBS (10 ml PBS/g brain tissue). After centrifuged at 10000 rmp for 10 min at 4°C, the supernatants were collected and were stored at -80°C. The total soluble protein in supernatants was used as the internal reference standard for the quantification of BDNF. They were determined by improved concentration BCA protein assay kits (Sangon Co., Ltd, Shanghai, China) and the BDNF enzyme-linked immunosorbent assay (ELISA) kits (Cusabio Co., Ltd, Wuhan, China) respectively.

The other half brain was used for immunohistochemical examination of brain BDNF in CA3 area of the hippocampus (see supplementary information). The other half brain was fixed in 10% neutral formalin and desiccated and embedded in paraffin. Subsequently, the tissues were cut into 5 μm thick serial sections. The sections including hippocampus area were selected for immunohistochemical examination for BDNF. After dewaxed by dimethylbenzene and hydrated by gradient alcohol, the sections washed by PBS for 3 times. Then the sections were treated with 3% hydrogen peroxide to block endogenous peroxidases for 20 min. After antigen repaired by microwave in 0.01 m citrate buffer solution, the sections were blocked with goat serum for 1 h at room temperature, then incubated at 4°C overnight with rabbit antibody against human BDNF (1: 200 dilution, Bioss Co., Ltd, Beijing, China). Afterwards, the sections were washed with PBS and subjected to immunohistochemical staining using a SP96000 kit (ZSGB Co., Ltd, Beijing, China). Next, the sections were washed with PBS and developed with 3,3’-diaminobenzidine (DAB) for 5 min. Finally the sections imaged under light microscopy.

▪ Data analysis

Data were analyzed by Spss11.5 software using one-way ANOVA followed by least significant difference test for the parameters among the different groups (two-tailed). Data were expressed as the mean ± S.E.M (standard error of the mean). The statistical significance was set at P<0.05.

Results

▪ Vanillin increased serum magnesium

Serum magnesium in the vanillin group was significantly higher than that of the untreated group and the fluoxetine group [F (3,43)=4.868, P=0.036; F (3,43)=4.868, P=0.016, respectively]. But, no obvious difference was found between the untreated group and the fluoxetine group. Serum magnesium in the fluoxetine group and the untreated group was still lower than the control group [F (3,43)=4.868, P<0.01; F (3,43)=4.868, P<0.01, respectively]. No obvious difference was found between the vanillin group and the control group.

▪ Brain BDNF was normalized by vanillin aroma

BDNF levels in both the vanillin and fluoxetine groups were significantly higher than that of the untreated group [F (3,43)=5.702, P=0.012; F (3,43)=4.703, P=0.014, respectively]. No obvious difference was found between the vanillin group and the fluoxetine group. When compared with the control group, BDNF in the untreated group was significantly lower [F (3,43)=5.702, P<0.01]. No similar result was found in the vanillin group and the fluoxetine group (Table 1).

Biochemical molecules

Vanillin
(n=13)

Fluoxetine
(n=13)

Untreated
(n=12)

Control
(n=8)

Serume [Mg2+]

0.983 ± 0.025*

0.871 ± 0.032##

0.885 ± 0.024##

1.035 ± 0.055

BDNF/Pr

0.524 ± 0.041*

0.520 ± 0.038*

0.371 ± 0.039##

0.644 ± 0.062

Note: The presented data are mean+SEM. Statistical analysis was carried out by one-way ANOVA among groups. *P<0.05 vs the untreated group; ##P<0.01 vs the control group.

Table 1: Serum magnesium and brain BDNF among groups after treatment.

By immunohistochemical staining, the expression of BDNF in the fluoxetine group, the vanillin group and the control group was strong. Relatively speaking, it was weak in the untreated group. At the same time, fewer neurons were observed in CA3 area in the untreated group. No similar result was found in the vanillin group (Figure 1).

Figure 1: Images of immuno histochemical examination of BDNF in CA3 area.
(×100)
A: CA3 area in the fluoxetine group; B: CA3 area in the vanillin group;
C: CA3 area in the untreated group; D: CA3 area in the control group.
BDNF was expressed strong in all groups except for the untreated group. Fewer neurons were observed in the CA3 area of the untreated group.

Discussion

Complementary therapies for the treatment of depression has revealed that odorant aroma can relieve depression and anxiety, restoring both physical and emotional well-being [8,9]. As an odorant, vanillin is generally rated as pleasant and correspondingly evokes positive moods [10]. It has even been used to combat depression by oral [11,12]. In our previous studies, we had demonstrated that vanillin could alleviate depressive symptoms in the depression model rats by elevating both serotonin and dopamine levels in brain tissue [7]. In present study, we would further explore other underlying mechanisms.

NMDA receptor antagonists and brain BDNF appear to be involved in the mechanism of depression [13]. NMDA receptor is a type of ionotropic glutamate receptors playing a unique role in synaptic functions. Persistent activation of NMDA receptor would lead to a large amount of calcium ions inflow, causing cytotoxicity and leading to depression. So, NMDA receptor antagonists, such as magnesium, may have antidepressant efficacy [14,15].

Magnesium is one of the most important elements and is involved in a number of biochemical processes crucial for the proper functioning of living organisms [16]. It also plays a vital modulatory role in brain biochemistry, influencing several neurotransmission pathways associated with the development of depression. Magnesium ions are inhibitors of NMDA receptor by competing with calcium ions and blocking the glycine site on the NMDA receptor [17]. A decline of magnesium is believed associated with ensuing neuronal cell death and subsequent functional impairment [18]. Magnesium deficiency increases the susceptibility of animals to stress and leads to many personality changes including depression [19,20]. On the contrary, supplementary of magnesium is demonstrated antidepressant-like effect [21,22]. Screening for compounds to increase neuronal magnesium concentration could be a promising instrument to identify new classes of antidepressants [13]. The antidepressant-like activity of magnesium involves the AMPA/BDNF pathway [23]. BDNF is linked to the pathophysiology of depression [24,25]. There is accumulating evidence for the role of BDNF in the pathophysiology of depression [26]. CUMS procedure induces cognitive impairments including depression as well as the reduction of BDNF level [27,28]. On the contrary, an intensified release of BDNF may mediate antidepressant effect via BDNF-TrKB signaling pathway [29,30]. In present study, after treatment for 4 w, vanillin aroma increased serum magnesium significantly compared with that of the untreated group. Increase of magnesium predicted the antidepressant effect of vanillin. Correspondingly, BDNF levels of the vanillin group were also increased remarkably which would mediate the antidepressant effect of magnesium by AMPABDNF pathway [23,31]. In the fluoxetine group, magnesium was not found increased significantly. However, fluoxetine itself also has an inhibitory effect on Ca2+-permeable AMPA receptors [32]. As a result, BDNF was also remarkably increased in the fluoxetine group, which was one of the mechanisms fluoxetine exerting the antidepressant effect [30]. We also noticed that the number of neurons in the CA3 were highly consistent with the level of brain BDNF (Figure 1). Fewer neurons were found in the untreated group since its BDNF level was lower. BDNF deficiency combined with a decrease in the number of neurons may be one of the reasons why depression cannot be recovered [33].

In present study, we investigated the underlying mechanisms of antidepressant effect of vanillin aromatherapy. Our results indicated that vanillin aromatherapy could increase serum magnesium, which elevated brain BDNF and mediated the antidepressant effect as NMDA receptor antagonists. Further investigations would be carried out around the related signaling pathway and receptors. The present evidences further support vanillin as a potential antidepressant by olfactory pathway.

Acknowledgments

This research was supported by National Natural Science Foundation of China (81000589).